Specificity of hydrolysis of bovine kappa-casein by cell envelope-associated proteinases from Lactococcus lactis strains

Abstract

The cell envelope-associated proteinases from Lactococcus lactis subsp. cremoris H2 (a P,-type proteinaseproducing strain) and SKII (a P111-type proteinase-producing strain) both actively hydrolyze the K-casein component of bovine milk but with significant differences in the specificity of peptide bond hydrolysis. The peptide bonds Ala-23-Lys-24, Leu-32-Ser-33, Ala-71-Gln-72, Leu-79-Ser-80, Met-95-Ala-96, and Met-106-Ala-107 were cleaved by both proteinase types, although the relative rates of hydrolysis at some of these sites were quite different for the two proteinases. Small histidine-rich peptides were formed as early products of the action of the cell envelope-associated proteinases on K-casein, implicating this casein as a possible significant source of histidine, which is essential for starter growth. The major difference between the two proteinase types in their action on K-casein was in their ability to cleave bonds near the C-terminal end of the molecule. The bond Asn-160-Thr-161 and, to a lesser extent, the bond Glu-151-Val-152 were very rapidly cleaved by the

Involvement of enzyme-substrate charge interactions in the caseinolytic specificity of lactococcal cell envelope-associated proteinases

“…3). As expected, the peptide consisting of residues 161 to 169 of -casein was not detected in the H2 P I -type proteinase digest of -casein since this proteinase has been shown previously not to hydrolyze the Asn-160OThr-161 bond (11). However, in the P IIItype proteinase digest of -casein, the fragment consisting of residues 161 to 169 was produced at a little under half the rate at which the corresponding peptide 1A-derived fragment 9-17 appeared.…”

“…Digestion of the -casein fragment of residues 153 to 169 by P I -and P III -type proteinases. A major distinction between P I -and P III -type proteinases in their specificity of hydrolysis of -casein lies in the resistance of the C-terminal region to hydrolysis by the P I -type proteinase as compared with the susceptibility of certain bonds in this region to P III -type proteinase-catalyzed hydrolysis (11,19). In particular, the Asn-160OThr-161 bond is hydrolyzed more rapidly than any other bond in -casein by the P III -type proteinase but is not detectably hydrolyzed by the P I -type proteinase (Fig.…”

“…However, in the P IIItype proteinase digest of -casein, the fragment consisting of residues 161 to 169 was produced at a little under half the rate at which the corresponding peptide 1A-derived fragment 9-17 appeared. This is not surprising since the proteinase must bind to several regions of the -casein molecule in addition to that involved during hydrolysis of the Asn-160OThr-161 bond (11,19), and thus, a lower overall rate of hydrolysis of this bond in -casein relative to that in peptide 1A would be expected. Digestion of the -casein fragment consisting of residues 98 to 111 and variants by P I -and P III -type proteinases.…”

“…The present study was designed to test the consistency of the proposed model with respect to the specificity differences of two types of lactococcal CEP in hydrolyzing -casein (11). The H2 and SK11 proteinases have been shown previously to be representative of the P I -and P III -type proteinases, respectively (12,13).…”

“…The H2 and SK11 proteinases have been shown previously to be representative of the P I -and P III -type proteinases, respectively (12,13). The specificity of action of these particular proteinases on ␣ s1 -, ␤-, and -casein has been well established (11)(12)(13), and they were selected for use in this study because of this. Oligopeptides corresponding to two regions of the -casein molecule which show different susceptibility to the two types of proteinase were synthesized.…”